Antibody scFv fragments without disulfide bonds made by molecular evolution.
We generated stable and functional cysteine-free antibody single-chain fragments (scFv) lacking the conserved disulfide bonds in both VH and VL. This was achieved by molecular evolution, starting from the scFv fragment of the levan binding antibody ABPC48, which is naturally missing one of the conserved cysteine residues, by using DNA shuffling and phage display. Several of the selected sequences were expressed and the resulting scFv proteins characterized by equilibrium urea denaturation. Three of the characterized proteins exhibit thermodynamic stability similar to the wild-type protein, and these cysteine-free mutant proteins can now be expressed in functional form in the Escherichia coli cytoplasm. We believe that such molecules are of great utility for use as intrabodies, can be produced by simpler expression strategies and may give further insight into the folding and stability of the immunoglobulin fold.
Proba
K
K
Biochemisches Institut, Universität Zürich, Switzerland.
Wörn
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A
Honegger
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Plückthun
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D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D003198:Computer Simulation; D017124:Conserved Sequence; D004220:Disulfides; D019143:Evolution, Molecular; D005803:Genes, Immunoglobulin; D006801:Humans; D007128:Immunoglobulin Fragments / Q000737:chemistry* / Q000235:genetics*; D007143:Immunoglobulin Heavy Chains / Q000737:chemistry / Q000235:genetics; D007147:Immunoglobulin Light Chains / Q000737:chemistry / Q000235:genetics; D007135:Immunoglobulin Variable Region / Q000737:chemistry* / Q000235:genetics*; D051379:Mice; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009194:Myeloma Proteins / Q000276:immunology; D017354:Point Mutation; D016133:Polymerase Chain Reaction; D011487:Protein Conformation
D004220:Disulfides; D007128:Immunoglobulin Fragments; D007143:Immunoglobulin Heavy Chains; D007147:Immunoglobulin Light Chains; D007135:Immunoglobulin Variable Region; D009194:Myeloma Proteins; C040835:myeloma protein A48, mouse